5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor α, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 μM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Symbiosis-specific expression of Rhizobium etli casA encoding a secreted calmodulin-related protein

Symbiosis between Rhizobium and its leguminous host requires elaborate communication between the partners throughout the interaction process. A calmodulin-like protein, termed calsymin, was identified in Rhizobium etli; a calmodulin-related protein in a Gram-negative bacterium had not been described previously. Calsymin possesses three repeated homologous domains. Each domain contains two predicted EF-hand Ca2+-binding motifs. Ca2+-binding activity of calsymin was demonstrated on purified protein. R. etli efficiently secretes calsymin without N-terminal cleavage of the protein. The gene encoding calsymin, casA, is exclusively expressed during colonization and infection of R. etli with the host. Expression of casA is controlled by a repressor protein, termed CasR, belonging to the TetR family of regulatory proteins. Mutation of the casA gene affects the development of bacteroids during symbiosis and symbiotic nitrogen fixation.

Role of the F-box protein Skp2 in lymphomagenesis

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G1-S transition by controlling the stability of several G1 regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Chloroplast-Targeted ERD1 Protein Declines but Its mRNA Increases during Senescence in Arabidopsis1

Arabidopsis ERD1 is a ClpC-like protein that sequence analysis suggests may interact with the chloroplast-localized ClpP protease to facilitate proteolysis. The mRNA encoded by the ERD1 gene has previously been shown to accumulate in response to senescence and to a variety of stresses and hormones. Here we show that the ERD1 protein, in contrast to the ERD1 mRNA, strongly declines in abundance with age, becoming undetectable in fully expanded leaves. Sequence analysis also suggests that ERD1 is chloroplast targeted, and we show in an in vitro system that the native protein is properly imported, processed, and present within the soluble fraction of the chloroplast, presumably the stroma. We show that ClpP protein, which is also present in the stroma, declines with age in parallel with ERD1. These results are consistent with the interaction of ERD1 and ClpP, but they suggest that it is unlikely that either plays a major role during senescence. Certain other chloroplast proteins decline with age coordinately with ERD1 and ClpP, suggesting that these declines are markers of an early age-mediated change that occurs within the chloroplast.

Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit α4

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the α4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged α4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of α4. Localization of Mid1 and α4 was influenced by one another in transiently transfected cells. Mid1 could recruit α4 onto microtubules, and high levels of α4 could displace Mid1 into the cytosol. Metabolic 32P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length α4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein–Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by α4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19ARF synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

Chromatin components as part of a putative transcriptional repressing complex

The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

CENP-E is an essential kinetochore motor in maturing oocytes and is masked during Mos-dependent, cell cycle arrest at metaphase II

CENP-E, a kinesin-like protein that is known to associate with kinetochores during all phases of mitotic chromosome movement, is shown here to be a component of meiotic kinetochores as well. CENP-E is detected at kinetochores during metaphase I in both mice and frogs, and, as in mitosis, is relocalized to the midbody during telophase. CENP-E function is essential for meiosis I because injection of an antibody to CENP-E into mouse oocytes in prophase completely prevented progression of those oocytes past metaphase I. Beyond this, CENP-E is modified or masked during the natural, Mos-dependent, cell cycle arrest that occurs at metaphase II, although it is readily detectable at the kinetochores in metaphase II oocytes derived from mos-deficient (MOS−/−) mice that fail to arrest at metaphase II. This must reflect a masking of some CENP-E epitopes, not the absence of CENP-E, in meiosis II because a different polyclonal antibody raised to the tail of CENP-E detects CENP-E at kinetochores of metaphase II-arrested eggs and because CENP-E reappears in telophase of mouse oocytes activated in the absence of protein synthesis.

Identification of a cellular receptor for subgroup E avian leukosis virus

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.

Convergent evolution of apolipoprotein(a) in primates and hedgehog

Apolipoprotein(a) [apo(a)] is the distinguishing protein component of lipoprotein(a), a major inherited risk factor for atherosclerosis. Human apo(a) is homologous to plasminogen. It contains from 15 to 50 repeated domains closely related to plasminogen kringle four, plus single kringle five-like and inactive protease-like domains. This expressed gene is confined to a subset of primates. Although most mammals lack apo(a), hedgehogs produce an apo(a)-like protein composed of highly repeated copies of a plasminogen kringle three-like domain, with complete absence of protease domain sequences. Both human and hedgehog apo(a)-like proteins form covalently linked lipoprotein particles that can bind to fibrin and other substrates shared with plasminogen. DNA sequence comparisons and phylogenetic analysis indicate that the human type of apo(a) evolved from a duplicated plasminogen gene during recent primate evolution. In contrast, the kringle three-based type of apo(a) evolved from an independent duplication of the plasminogen gene approximately 80 million years ago. In a type of convergent evolution, the plasminogen gene has been independently remodeled twice during mammalian evolution to produce similar forms of apo(a) in two widely divergent groups of species.

A cdc5+ homolog of a higher plant, Arabidopsis thaliana

We cloned and characterized a cDNA corresponding to a cdc5+ homolog of the higher plant, Arabidopsis thaliana. The cDNA, named AtCDC5 cDNA, encodes a polypeptide of 844 amino acid residues. The amino acid sequence of N-terminal one-fourth region of the predicted protein bears significant similarity to that of Schizosaccharomyces pombe Cdc5 and Myb-related proteins. Overexpression of the AtCDC5 cDNA in S. pombe cells is able to complement the growth defective phenotype of a cdc5 temperature-sensitive mutant. These results indicate that the AtCDC5 gene is a plant counterpart of S. pombe cdc5+. This is the first report of a cdc5+-like gene in a multicellular organism. We also demonstrated that a recombinant AtCDC5 protein possesses a sequence specific DNA binding activity (CTCAGCG) and the AtCDC5 gene is expressed extensively in shoot and root meristems. In addition, we cloned a PCR fragment corresponding to the DNA binding domain of human Cdc5-like protein. These results strongly suggest that Cdc5-like protein exists in all eukaryotes and may function in cell cycle regulation.

The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.